Our interest in cellular stress resistance
emerged from the finding that fibroblasts derived from young adult
Snell dwarf mice were resistant to multiple forms of cytotoxic stress.

Stress resistance in mouse primary
fibroblast cultures

Mutations in worms and flies that lead to increased life span very often
also endow these invertebrates with high levels of resistance to several
forms of cellular stress, including ultraviolet irradiation, heat exposure,
and oxidation stress. To see if mutations that increase mouse
longevity also lead to cellular stress resistance, we evaluated stress
resistance in vitro of fibroblast cell lines derived from the skin of
Snell dwarf mice, in which a mutation at the
Pit-1 gene leads to endocrine defects, small body size, and a 40% increase
in mean and maximal life span. The results showed [PubMed]
that fibroblasts from dwarf mice were resistant to cell death induced by
each of five different stress agents: heat, UV light, hydrogen peroxide,
paraquat, and the heavy metal cadmium. This finding has
several implications. First, it suggests that the long life span and
exceptional disease resistance of the dwarf mice may be due to an intrinsic
resistance, at the cellular level, to multiple forms of stress.
Second, it implies that stress resistance of fibroblasts, and perhaps also
other cell types, can be modulated by early life exposure to varying levels
of hormones including IGF-I and thyroid hormones, and that the mechanism of
resistance is epigenetic, i.e. related to alterations in gene expression
patterns that survive multiple rounds of mitosis in vitro. Third, it
suggests that the relationships among hormonal patterns, organismic
longevity and cell stress resistance may have emerged very early in
eukaryote evolution, i.e. prior to the divergence between flies, worms, and
mammals.

Follow-up studies have added useful detail to this initial picture.
(1) Similar stress resistance profiles were seen using fibroblasts from two
other kinds of long-lived mutant mice, the Ames dwarf, which also has
multiple endocrine abnormalities, and the growth-hormone receptor KO mouse,
which has resistance to GH action and therefore low IGF-I levels.
These findings show that the stress resistance effect is not limited to a
single mutation, single colony, or single background stock. [PubMed].
(2) Resistance of these cells to the DNA damaging agent MMS, and to UV
light, suggests that the cells are able to fend off both oxidative and
non-oxidative injuries. (3) The difference in stress resistance
between cells from Snell dwarf and control cells is not apparent in the
first week of life, but develops in the next few months, presumably as a
consequence of cellular differentially in an unusual hormonal environment [PubMed].
(4) Cells from mice whose aging rate has been slowed by caloric restriction
or methionine deprivation do not show a similar resistance in vitro,
suggesting that the mechanisms of lifespan extension induced by these diets
may differ from those involved in the dwarf mice [PubMed].

Resistance to oxygen-mediated growth crisis

Campisi and her collaborators have shown that the growth "crisis"
typically seen after 5 - 15 doublings in cultured mouse embryonic
fibroblasts (MEF), characterized by apoptosis, cessation of net cell
production, aneuploidy, and transformation, represents a toxic response to
the oxygen levels (20%) used in standard cell cultures. These workers
found that culturing MEF at a level of oxygen (~3%) closer to the
concentration of post-capillary tissue beds, prevents growth crisis and
diminishes the rate of accumulation of mutations and aberrations thought to
represent early stages in cellular transformation. We have found [PubMed] that
fibroblasts from normal adult skin also exhibit a decline in cell
accumulation at passages 5 - 10, which can be delayed considerably by growth
in 3% oxygen. Cells from Snell dwarf donors, however, resist these changes
in culture growth patterns even when grown at 20% oxygen. This
suggests that resistance to oxygen-mediated genetic alterations might
contribute to the low age-adjusted tumor incidence in Snell dwarf mice.

Metabolic abnormalities: rotenone and low glucose

In addition to their resistance to death induced by lethal agents,
fibroblasts from Snell dwarf mice show resistance to metabolic abnormalities
induced by two conditions: exposure to low doses of rotenone, and culture in
medium with very low glucose concentrations. Normal fibroblasts are
able to reduce extracellular substrates, like the tetrazolium dye WST-1,
through a poorly characterized set of plasma membrane proteins and
intermediate electron acceptors, known collectively as the Plasma Membrane
Redox System or PMRS. Fibroblast reduction of WST-1 is diminished,
through unknown pathways, by exposure to rotenone (better known as an
inhibitor of the mitochondrial electronic transport chain Complex I), or by
culture in medium containing very little glucose. PMRS activity in cells
from Snell dwarf donors, however, is resistant both to rotenone and to low
glucose media. When cells from normal (non-dwarf) mice are evaluated,
those individual cell lines that best resist the effects of low glucose
media are most resistant to the lethal effects of peroxide and cadmium,
suggesting that the pathways that regulate death after lethal stress and
modulate PMRS action in low glucose conditions share overlapping features [PubMed].
Further work on the molecular basis for resistance to low glucose medium and
to rotenone may provide new insights into the basis for resistance of Snell
dwarf cells to lethal injury as well.

Cell lines from long-lived species of rodents

To see if the mechanisms that protect Snell dwarf cells from lethal
stresses, and from metabolic inhibitors, might play any role in the
evolutionary processes that generate long-lived species of rodents, we
prepared multiple cultures of skin-derived fibroblasts from mice, rats,
deermice, white-footed mice, red squirrels, fox squirrels, porcupine,
beavers, and little brown bats. There was a clear correlation between
longevity of the species and the ability of their cultured cells to resist
lethal injury from hydrogen peroxide and cadmium [PubMed]. Resistance to heat
and to MMS was also associated with species lifespan to varying degrees,
depending on the assumptions of the statistical analysis. Cells from
long-lived rodents were not, however, more resistant to death induced by paraquat or by UV light. These results suggest that evolution of
long-lived rodents (and perhaps long-lived species in other orders?) may
require adjustment of cellular resistance to some, but not to all, forms of
lethal injury.

Follow-up studies: work in progress

These discoveries open up a wide range of questions for future studies:

Are other cell types in dwarf mice, including hepatocytes and
skin cells, also relatively resistant to multiple forms of stress?

Do differences in cellular stress resistance among genetically
heterogeneous mice also predict life span and resistance to multiple
diseases?

What is the molecular basis for differences in stress resistance
between normal and dwarf mice?

What changes in hormonal milieu lead to differential stress resistance
in dwarf mice, and can this property be modulated by endocrine factors in
adult animals?

Do differences in stress resistance contribute to differences in life
span between long- and short-lived species?

Do non-genetic approaches to longevity extension, such as caloric
restriction, also increase cellular stress resistance?